Purification of coumestrol



United States Patent Qfidce Patented July 21, 1959 PURIFICATION OFCOUMESTROL Oliver H. Emerson, Orinda, and Emanuel M. Bickolf, Berkeley,Calif., assignors to the United States of America as represented by theSecretary of Agriculture No Drawing. Application April 4, 1958 SerialNo. 726,588

5 Claims. (Cl. 260-3433) (Granted under Title 35, US. Code (1952), see.266) A non-exclusive, irrevocable, royalty-free license in the inventionherein described, throughout the world for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention relates to and has as its prime object the provision ofnovel methods for preparing coumestrol. A particular object of theinvention concerns the provision of novel methods for preparing purecrystalline coumestrol from impure preparations containing coumestrol.Further objects and advantages of the invention will be evident from thedescription herein.

In the co-pending patent application of E. M. Bickoff and A. N. Booth,Serial No. 693,785, filed Oct. 31, 1957, there is disclosed a previouslyunknown compound and methods for isolating it from forage crops such asladino clover, alfalfa, etc. The inventors also disclose that thecompound exhibits estrogenic activity and may be employed in animalraising to obtain increased weight gain and increased efliciency of feedutilization. The compound in question has been named coumestrol by theinventors and structurally it is 7,6-dihydroxycoumarino-(3',4-3,2)-coumarone having the formulawherein each of the radicalsrepresents an acyl radical. The acyl radicals may be the same ordifferent from one another and may be derived from any carboxylic acid;for example, acetic acid, propionic acid, butyric acid, isobutyric acid,valeric 2 acid, isovaleric acid, caproic acid, caprylic acid, capricacid, lauric acid, myristic acid, palrnitic acid, stearic acid, oleicacid, palmitoleic acid, benzoic acid, dinitrobenzoic acid, orthotoluicacid, metatoluic acid, paratoluic acid, etc.

The hydrolysis of the coumestrol ester essentially involves contactingit with an alkali metal hydroxide such as potassium hydroxide, sodiumhydroxide, an alkali metal alcoholate, or the like. Since the coumestrolesters are relatively insoluble in Water, the reaction is prefera blyconducted in solution in an inert organic solvent such, as methanol,ethanol, propanol, isopropyl alcohol, any of the isomeric butylalcohols, the mono-methyl or monoethyl ethers of ethylene glycol, etc.An excess of alkali over the theoretical amount necessary to saponifythe two acyloxy radicals is generally employed. Usually there is usedsufficient alkali to furnish about six or more moles of OH- per mole ofester. The temperature of reaction is not critical and may be variedfrom about 0 to 100 C. Usually, a temperature of about 50-75 C.

. is preferred as giving a rapid rate of reaction Without danger ofdecomposition. The reaction is complete when the two acyloxy groups havebeen converted to hydroxy groups.

After the alkaline hydrolysis is complete, the reaction mixture isacidified by adding an acid such as hydrochloric, sulphuric, phosphoric,or the like. This converts the salt (or phenolate) form of the productinto the free phenol form of coumestrol. It is also believed that in thealkaline hydrolysis the lactone ring of the coumarin moiety is opened.However, acidification of the hydrolysis product restores the originallactone formation.

Coumestrol is readily isolated from the acidified reaction mixture onaddition of water since it is relatively insoluble in water. Thecoumestrol so obtained may be re-crystallized from methanol,methanol-chloroform, or acetone. Prior to recrystallization, it isusually desirable to treat the product to remove colored impurities.This may conveniently be done by contacting the coumestrol in solutionin acetone, methanol, or the like with charcoal. The charcoal absorbsthe impurities so that coumestrol recrystallized from the solution isespecially light in color and pure.

It is often preferred to add an antioxidant to the reaction mixture inthe alkaline hydrolysis described above for the purpose of preventingoxidation of coumestrol during the reaction. For this purpose one mayemploy any of the antioxidants generally useful for preventing oxidationof fats, oils, rubber, etc. Typical antioxidants V which may be employedare nordi'hydroguaiaretic acid;

.' used, i.e., about 0.01 to 1%, based on the weight ofcoumestrol ester.A greater proportion of antioxidant of course, can be employed butgenerally without any particular advantage. Usually it is preferred toemploy antioxidants which are nitrogenous bases and which formwater-soluble salts with acids. In such case the antioxidant remains insolution when the alkaline hydrolysis mixture is acidified, whereas thecoumestrol product precipitates out of solution. For this reason it ispreferred to use 2,2,4-trimethyl-1,2-dihydroquinoline or its variousderivatives thereof such as the 6-ethoxy, 6-phenyl, or 7- chloroderivatives. However, even if one uses an antioxidant which precipitateswith the coumestrol, no harm is done because the accompanyingantioxidant will serve to stabilize the coumestrol on storage. Inaddition to, or in place of, the antioxidant one may minimize oxidationduring the alkaline hydrolysis by conducting the reaction in anatmosphere of an inert gas such as nitrogen.

An especially important aspect of the invention is that it may beutilized for the production of pure crystalline coumestrol from mixturescontaining the same. One problem in the production of coumestrol, eitherby isolation from natural sources or by organic synthesis is that it isdifficult to obtain this compound in pure form. This is particularly thecase because coumestrol do% not crystallize readily especially whencontaminated with significant amounts of impurities. As a consequence itis difficult to isolate coumestrol from associated impurities. Thisproblem is demonstrated by reference to the co-pending application of O.H. Emerson and E. M. Bickoff, Ser. No. 710,586, filed Jan. 22, 1958,wherein is described and claimed methods for synthesizing coumestrol.The method, briefiy described, involves reacting an omega(-2,4-dialkoxyphenyl) resacetophenone with an alkyl' halo.- formate andhydrolyzing the ester product to produce a3(2,4-dialkoxyphenyl)-4,7-dihydroxy coumarin. This. compound is thenheated with an aromatic amine hydro.- halide to produce coumestrol. Theinventors point out that to avoid the difficulty of isolatingcoumestrol, it is converted to an ester and recovered in the ester form.

It has now been found that coumestrol can be obtained in purecrystalline form from impure preparations thereof by a process whichessentially involves subjecting the crude preparation to reaction withan acylating agent, separating the coumestrol ester from the acylation.prodnet and hydrolyzing the coumestrol ester tov obtain purecoumestrol.

In applying this purification process the crude coume-- strolpreparation may be one produced by any means, for example, by theheating of a 3(2,4-dialkoxyphenyl)-4;7- dihydroxy coumarin with anaromatic amine hydrohal'id'e in accordance with the Emerson and Bickoffapplication or by extraction from forage materialsby the procedures ofthe aforesaid Bickofi and Booth application.

In the first step of the process of this invention the crude coumestrolpreparation is reacted with an acylating agent. The acylating agent maybe, for example, the anhydride, chloride, bromide, or iodide of anyaliphatic and aromatic hydrocarbon carboxylic acid such as acetic,propionic, butyric, isobutyric, valeric, isovaleric, caproic, caprylic,capric, lauric, myristic, palrnitic, stearic, arachidic, oleic,palmitolei'c, benzoic, dinitro-benzoic, otoluic, m-toluic, p-toluic,etc. Generally it is preferred to employ a low molecular weightacylating agent such as acetic anhydride or propionic anhydride. It isobvious. that by selection of the acylating agent, any desiredcoumestrol ester can be prepared; The acylating agent is employed in aproportion to furnish at least 2 moles, of the acyl radical per mole ofcoumestrol thus toensure esterifying both hydroxyl groups. To enhancecontact between the reactants an inert solvent may be added, or, in thealternative, such an excess of acylating agentmay be used to, functionboth as reactant and solvent. Suitable inert solvents are, for example,benzene, toluene, xylene, dioxane, acetone, ether, methyl-ethyl ketone,di-

ethylketone, di-isopropyl, ether, the diethyl ether, of; ethyl:

ene glycol, etc. The temperature of reaction is not critical and may bevaried from about 0 to 175 C. Usually temperatures of around to 150 C.are used to expedite the esterification. The reaction is of coursecomplete when the desired coumestrol di-ester is formed. To expedite theesterification, it is generally preferred to add to the reaction systema conventional esterification promoter such as for example the alkalisalt of the acid in question (e.g., sodium acetate when coumestroldiacetate is being prepared), pyridine, quinoline, N-dimethyl aniline,potassium carbonate, or the like. The coumestrol esters are readilyisolatable from the reaction mixture on addition of water since they arerelatively insoluble in this solvent. Moreover the coumestrol esters canbe readily isolated in pure form since they crystallize easily. Thusthey may bev crystallized from such solvents as acetonitrile, acetone,acetone-alcohol, etc. Moreover, the lower aliphatic esters of coumestrolcan be isolated by sublimation. For example coumestrol diacetatesublimesat about 150 C. at a pressure of 10- mm. of mercury.

After separation of the coumestrol ester from the esterificationreaction mixture, the ester is hydrolyzed as previously described toproduce pure crystalline coumestrol. This pure coumestrol is a highlydesirable product as being free from impurities it will exert desiredphysiological results. without interference or complications as might beexperienced with an impure product.

As disclosed in the aforementioned patent application of Bickofli andBooth, coumestrol exhibits estrogenic properties and is useful in animalraising to increase growth rate and to increase efficiency of feedutilization. As a typical example, coumestrol may be incorporated inminor amounts, for example 0.001 to 1 lb. per ton, in conventional feedcontaining a major proportion of vegetable material such as corn, wheat,barley, milo, alfalfa, cottonseed meal, soybean meal, etc. Suchsupplemented feed when supplied to animals particularly those grown formeat purposes, e.g., steers, will cause the animals to gain weight morerapidly and to produce more flesh per pound of feed.

The invention is further demonstrated by the following examples whereinExample I illustrates the synthesis of impure coumestrol according tothe process of the aforesaid Emerson and Bickoff application andExamples II and III illustrate the preparation of pure coumestrol inaccordance with the present invention.

Example I Omega (2,4-dimethoxyphenyl) resacetophenone (3.1 g.), acetone(50 ml.), methyl chloroformate (2 ml.), and potassium carbonate (8 g.)were refluxed for four hours. The reaction mixture was cooled, dilutedwith ml. of water and acidified with hydrochloric acid. The precipitatewas filtered, washed with water and dried. The dry precipitate wasdissolved in about 20 ml. of methanol and to this solution was addedsufiicient of a 20% solution of, potassium hydroxide in methanol to turnalizarinyellow orange (pH about 11). The solution was refluxed for 10minutes under an inert atmosphere, then cooled, diluted with water, andacidified with hydrochloric acid. The precipitate was filtered off andrecrystallized from alcohol. The product,3(2,4-dimethoxyphenyl)-4,7-dihydroxy coumarin, obtained in a yield of75%, had a melting point of 250-255 C. Analysis-Calculated for C H O -C,64.95%; H, 4.49%. Found: C, 65.0%; H, 4.5%

One gram of 3(2,4-dimethoxyphenyl)-4,7-dihydroxy coumarin was mixed with2 grams of aniline hydrochloride. The mixture was heated under anatmosphere of carbon dioxide for 3.5 hours at 210220 C. The reactionmixture was cooled, then successively extracted with water (about. 10cc.) then with alcohol (about 5- cc.) toremove pigmented. impurities.The residue was an impure preparation ofvcoumestrol obtained in a yieldQf.'60.%.

Example 11 The crude coumestrol (530 mg.) obtained as described inExample I, was suspended in a mixture of 5 cc. acetic anhydride and 0.5g. fused sodium acetate. The mixture was boiled for 5 minutes thenpoured into water. The coumestrol diacetate was filtered off andrecrystallized from acetonitrile. The recrystallized product had amelting point of 234235 C. Analysis.-Calculated for C H O C, 64.75; H,3.44%. Found: C, 64.7; H, 3.51%. The yield was 55%, based on the amountof 3(2,4-dimethoxyphenyl) 4,7 dihydroxy coumarin. The product may alsobe termed 7',6-diacetyl0xy coumarino- (3',4'-3,2)-coumarone.

One hundred mg. of the coumestrol diacetate was added to cc. of 10%methanolic potassium hydroxide and to this mixture was added one drop of6-ethoxy-2,2,4- trimethyl-1,2-dihydroquinoline as an antioxidant. Thevessel containing the mixture was flushed out with nitrogen and themixture was refluxed for a half hour. The refluxed solution was thencooled and enough hydrochloric acid added to make the solution stronglyacid. An equal volume of water was then added to precipitate thecoumestrol. The precipitate was filtered off and recrystallized fromacetone to give a 90% yield of pure, crystalline coumestrol. Testing ofthe product by chromatographic methods and by ultra-violet spectroscopyindicated it was completely free from impurities.

Example III Coumestrol diacetate (100 mg.) was refluxed 40 minutes with10 ml. of 6% methanolic potassium hydroxide in an atmosphere ofnitrogen. The mixture was then cooled, diluted with an equal volume ofwater, acidified with dilute hydrochloric acid, and concentrated in astream of carbon dioxide to precipitate the coumestrol. The coumestrolwas separated, dissolved in acetone-methanol and boiled a few minuteswith about 20 mg. of

6 charcoal. After removal of the charcoal, the solution was concentratedto about 3 ml. and allowed to stand for about an hour. The purecoumestrol which crystallized out of solution was then separated.

Having thus described the invention, what is claimed is:

1. The method of recovering pure 7',6-dihydroxycoumarino-(3',4'-3,2)coumarone from an impure mixture containing the same which comprisesreacting the mixture with an acylating agent of the group consisting ofthe anhydride, the chloride, the bromide, and the iodide of a carboxylicacid selected from the group consisting of alkanoic acids, oleic acid,palrnitoleic acid, benzoic acid, dinitrobenzoic acid, o-toluic acid,m-toluic acid, and p-toluic acid, to produce the corresponding ester ofsaid 7,6-dihydroxycoumarino-(3,4'-3,2) coumarone, separating said esterfrom the esterification reaction mixture, subjecting it to alkalinehydrolysis to convert the ester groups to hydroxy groups, acidifying thealkaline reaction mixture to produce pure 7,6-dihydroxycoumarino-(3',4-3,2) coumarone, and isolating the pure7',6-dihydroxycoumarino-(3',4'-3,2) coumarone from the resultingacidified mixture.

2. The method of claim 1 wherein the acylating agent is aceticanhydride.

3. The method of claim 1 wherein potassium hydroxide is used as thealkaline agent for carrying out the alkaline hydrolysis.

4. The method of claim 1 wherein the alkaline hydrolysis is conducted inthe presence of an antioxidant.

5. The method of claim 1 wherein the alkaline hydrolysis is conducted inthe presence of a minor proportion of6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline as an antioxidant.

References Cited in the file of this patent Karrer: Organic Chem, 4thEnglish ed., p. 431, Elsevier Pub. Co., New York (1950).

1. THE METHOD OF RECOVERING PURE 7'',6-DIHYDROXYCOUMARINO-(3'',4''-3,2)COUMARONE FROM AN IMPURE MIXTURE CONTAINING THE SAME WHICH COMPRISESREACTING THE MIXTURE WITH AN ACYLATING AGENT OF THE GROUP CONSISTING OFTHE ANHYDRIDE, THE CHLORIDE, THE BROMIDE, AND THE OIDIDE OF A CARBOXYLICACIDS SELECTED FROM THE GROUP CONSISTING OF ALKANOIC ACIDS, OLEIC ACID,PAMITOLETIC ACID, BENZOIC P-TOLUIC ACID, TO PRODUCE THE CORRESPONDINGESTER OF SAID 7'',6-DIHYDROXYCOUMARINO-(3H,4''-3,2) COUMARONE,SEPARATING SAID ESTER FROM THE ESTERFICATION REACTION MIXTURE,SUBJECTING IT TO ALKALINE HYDROLYSIS TO CONVERT THE ESTER GROUPS TOHYDROXY GROUPS, ACIDIFYING THE ALKALINE REACTION MIXTURE TO PRODUCE PURE7'',6-DIHYDROXYCOUMARINO-(3'',43,2) COUMARONE, AND ISOLATING THE PURE7'',6-DIHYDROXYCOUMARINO-(3'',4''-3,2) COUMARONE FROM THE RESULTINGACIDIFIED MIXTURE.